Acidic ionic liquid modified silica gel for adsorption and separation of bovine serum albumin (BSA)

Abstract

In this work, silica gel (SiO₂) was modified with an acidic ionic liquid (Im⁺·HSO₄⁻) and the product was characterized using infrared spectroscopy, thermogravimetric analysis and elemental measurement. Then it was used in the adsorption of bovine serum albumin (BSA) for the first time. Then the influences of initial concentration, temperature, solid–liquid ratio and pH were investigated. These studies showed that the adsorption equilibrium data fit well with the Langmuir model and the pseudo-second-order model could be used to describe the adsorption kinetics more accurately. The major thermodynamic parameters including the change of enthalpy, entropy and Gibbs free energy indicated that the adsorption was an exothermic and spontaneous process, which was controlled by chemical and physical interactions. The mechanism was also investigated using characterization and comparative experiments. All of the proteins tested reached their maximum adsorbed amount on SiO₂·Im⁺·HSO₄⁻ when the pH was around their isoelectric point, and the adsorption capacity was lowered with the increase of protein volume. After the desorption using an aqueous solution of sodium chloride, the SiO₂·Im⁺·HSO₄⁻ could be recycled for several times. Finally, the selective adsorption of BSA and bovine hemoglobin from cow blood could be successfully realized under the appropriate pH conditions. This study is aimed to provide a preparative method for the selective adsorption and separation of BSA with a supported ionic liquid. Furthermore, it is expected to enrich the separation materials of proteins and extend the potential functions of ionic liquids.

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1. Introduction

At the moment, the adsorption of protein has become a very important and active field, which is attracting great attention from academia and industry. With the rapid progress of science and technology, the development of new types of adsorption and separation material is also more urgent. Common adsorption media for proteins can be divided into metallic biomaterials, ceramic biomaterials, polymeric biomaterials, composite biomaterials, hybrid biomaterials, and so on. The adsorption process of protein on metallic materials is relatively complex, and the interaction between protein and metal may lead to denaturation of the former. So more and more new polymer sorbents have been designed and prepared in the last decade. The common polymer material for protein adsorption is based on poly(ethylene glycol) polymer,¹ poly(vinyl pyrrolidone),² poly(vinyl alcohol) (PVA),³ zwitterionic polymers⁴ and complex polysaccharide,⁵ and so on. However, more emphasis has been put on the novelty of structure and high adsorption capacity and most of these polymers have a complex framework and preparation process, however, their reusability and practicability need to be further improved. Therefore, chemists are continuously focusing on the development of efficient, convenient, reliable and economical adsorbents for the separation of proteins.

In recent years, immobilized ionic liquids (IL) have been popularly applied in the field of separation science, and there are some successful examples in the separation of small bioactive molecules.^6,7 So some researchers began to consider its application with biological macromolecules. Yuan et al.⁸ used an IL polymeric monomer, 1-vinyl-3-butylimidazolium chloride (ViBuIm⁺·Cl⁻) to synthesize a new macroporous polymer which was successfully applied in the adsorption of five typical proteins. A nanocomposite was found to have selectivity towards acidic proteins, which was prepared by the copolymerization of 1-vinyl-3-ethylimidazolium bromide (monomer) and 1,4-butanediyl-3,3′-bis-L-vinylimidazolium dibromide (crosslinker) to encapsulate silica (SiO₂) nanospheres.⁹ Furthermore, in order to solve the potential difficulty of recovery for the tiny particles (nm level), magnetic nanoparticles modified with 1-hexyl-3-methylimidazolium bromide (HmIm⁺·Br⁻) were invented for the adsorption of proteins at various pH, which could be easily separated from the system by using a magnet.¹⁰ Similarly, Im⁺·Cl⁻ was immobilized on the surface of iron(II,III) oxide (Fe₃O₄) nanoparticles to capture hemoglobin.¹¹ However, the commercial magnetic nanoparticles are more expensive than ordinary supports (such as SiO₂ gel) and self-made ones will impose an additional burden on the sorbent preparation. Even if there is no external magnetic field, the operating mode of solid-phase extraction or column chromatography can also avoid the difficulty of recovery for the previously described sorbents. Furthermore, there are few applications reported using immobilized IL with other anions except for halogen anions [bromide (Br⁻) and chloride (Cl⁻)] up to now. More IL are needed for the exploration of their interaction mechanism with protein macromolecules.

Bovine serum albumin (BSA) is a globulin with two peptide chains comprising 583 amino acid residues (RCSB Protein Data Bank). Among them 35 cysteines form 17 disulfide bonds, and there is a free thiol at the 34^th position of the peptide chain. Bovine hemoglobin (BHb) is its homologous protein in the blood of cows. In previous research, electrophoresis, focusing electrochromatography¹² and molecularly imprinted gel polymers¹³ have been used to separate them for analytical or preparation purposes. In this work, imidazolium hydrogen sulfate (Im⁺·HSO₄⁻) was used to modify the particle surface of common SiO₂, and the product was characterized using elemental measurements, electron microscopy, infrared spectroscopy (IR), thermogravimetric analysis (TGA), and pore and specific surface size analysis. Then use of Im⁺·HSO₄⁻ was explored for the appropriate adsorption and desorption conditions for BSA on the synthesized sorbent, and the adsorption mechanism was observed using detailed characterization, isotherms, kinetics thermodynamics and comparative studies. Adsorption kinetics are especially, important and necessary because it is helpful not only to understand the adsorption behaviors but also to explore the interaction mechanism between IL and proteins. Various kinetic models indicate that the process is physical adsorption, chemical adsorption or mixed mode adsorption, and the latter is supposed to be the possible mechanism used this study. Finally, the selective adsorption for BSA and BHb on the IL-modified SiO₂ was also investigated and discussed. As basic research, the aim of this work is to provide a preparative method for the adsorption and selective separation of BSA using a supported IL sorbent. However, its use is also expected to enrich the separation materials for proteins and extend the potential functions of IL.

2. Experimental

2.1. Materials and reagents

For the synthesis of Im⁺·HSO₄⁻-modified SiO₂, commercial SiO₂ (pore size: 80–100 Å) was obtained from the Hailang Silica Gel Desiccant Factory (Qingdao, China). N-Methyl imidazole was provided by the Xishi Chemical Technology Co., Ltd. (Shanghai, China), and 3-chloropropyl trimethoxysilane was purchased from the Wanda Chemical Co., Ltd. (Shandong, China). Dichloromethane, ether, toluene, acetone, nitric acid (HNO₃) and concentrated sulfuric acid (H₂SO₄) were obtained from the Kelong Chemical Reagent Factory (Chengdu, China). All the involved protein standards were all purchased from Sigma-Aldrich Co. Ltd. (St. Louis., MO, USA), and the fresh sample of cow blood was obtained from a suburban slaughterhouse in Chengdu city, China. The previous reagents and solvents were of analytical reagent grade or higher. Experimental deionized water (H₂O) was purified using the Milli-Q water system with a 0.4 mm filter (Millipore Corp., Bedford, MA, USA) before use.

2.2. Apparatus

The elemental content of Im⁺·HSO₄⁻-modified SiO₂, were determined using a EuroEA3000 elemental analyzer (EuroVector Instruments and Software, Milan, Italy). The micro morphology was observed using JSM-7500F scanning electron microscope (SEM; Jeol, Tokyo, Japan) operated at 5.0–10.0 kV and a Tecnai G2 F20 S-TWIN transmission electron microscope (TEM; FEI, Hillsboro, OR, USA) operated at 20.0–50.0 kV, and the samples were coated with gold using the sputtering technique. In order to determine characteristic functional groups on the sorbent, Fourier transform infrared spectra (FT-IR) were recorded on an IR L1600300 spectroscope (PerkinElmer, Fremont, CA, USA) in the range of 4000–400 cm⁻¹ with potassium bromide (KBr) pellets. TGA was performed using a TG 209 F1 Iris instrument (NETZSCH-Gerätebau GmbH, Selb, Germany) with a nitrogen (N₂) heating rate of 10 °C min⁻¹ from 30 °C to 80 °C. In the determination of N₂ adsorption–desorption isotherms, a Nova 1000e surface area and pore size analyzer (Quantachrome Instruments, Boynton Beach, FL, USA) and a SSA-3500 specific surface area analyzer (Builder Company, Beijing, China) were used under vacuum.

2.3. Preparation of IL modified SiO₂

The target IL modified SiO₂ was prepared (shown in Fig. 1) on the basis of our previous study.¹⁴ In order to increase reactive groups on the particle surface of the support material, the commercial SiO₂ was firstly activated using HNO₃–H₂O (1 : 1, v/v) and then refluxed for 8 h. The resulting mixture was filtered and washed thoroughly with firstly deionized H₂O and then acetone. The mixture was dried under vacuum at 60 °C for 12 h before chemical surface modification. N-methyl imidazole was firstly mixed with equimolar 3-chloropropyl trimethoxysilane and refluxed under N₂ protection at 120 °C for 24 h. The product was washed with diethyl ether and dissolved in toluene, and concentrated H₂SO₄ was added dropwise and then the mixture was reacted at 120 °C for 15 h to replace the Cl⁻ with HSO₄⁻ completely. After the anion exchange, the product was dissolved in anhydrous toluene and reacted with an equivalent amount of activated SiO₂ gel under stirring at 120 °C for 24 h. The resulting product of SiO₂·Im⁺·HSO₄⁻ was obtained after filtration and washing (for three times) successively with dichloromethane and acetone, and then it was dried under vacuum at 60 °C before characterization and further use.

Fig. 1 Synthetic process of SiO₂·Im⁺·HSO₄⁻.

2.4. Adsorption and desorption experiments

The experiments in the investigation of SiO₂·Im⁺·HSO₄⁻ for its adsorption efficiency, capacity, kinetics, and isotherms and thermodynamics were performed as follows: a specific amount of synthesized sorbent was placed in an 100 mL Erlenmeyer flask and then mixed with 20 mL of an aqueous solution of BSA of a certain concentration. The flask was thoroughly shaken for a specified time in a water bath shaker (100 rpm) at a certain temperature. After that, the mixture was centrifuged for 5 min at 3000 rpm, and then the supernatant was sampled and the ultraviolet-visible (UV-vis) absorbance was measured. The protein contents were all determined on a TU-1810 UV-vis spectrometer (Purkinje General Instrument Co., Ltd., Beijing, China). Using the Lowry method,¹⁵ the working calibration curve of BSA was prepared using deionized H₂O as a blank control at 750 nm. The linear regression equation was y = 0.0200x + 0.0579, where y and x were the values of absorbance and sample concentration (μg mL⁻¹), respectively. The calibration curve in the study showed good linearity (correlation coefficient (R²) = 0.9990) over the tested concentration range (0–600 μg mL⁻¹). After the adsorption process, the amount of unadsorbed protein in the supernatant could be calculated using the calibration curve that was obtained. Finally, the adsorption efficiency (E, %) was calculated using eqn (1) and the adsorption capacity (q_e, mg g⁻¹) was obtained using eqn (2), as follows:where C₀ and C₁ are the concentrations (mg L⁻¹) of protein before and after adsorption on IL modified SiO₂, respectively; V represents the volume (L) of protein solution and m is the mass (g) of the sorbent. Uncertainty of E = ±0.4% and uncertainty of q_e = ±0.5 mg g⁻¹.

In the desorption study, an appropriate amount of protein loaded sorbent was mixed with a 6% aqueous solution of sodium chloride (NaCl) in a 50 mL Erlenmeyer flask with agitation of 150 rpm. The released protein concentration in the supernatant was also determined using the calibration curve developed as described previously. The desorption efficiency (D, %) of protein is described by eqn (3):

where C₀, C₁ and C₂ are the concentrations (mg L⁻¹) of protein in the initial solution, the supernatant and desorption solution, respectively; V₀ and V₂ represent the volumes (L) of the initial solution and eluent, respectively.

3. Results and discussion

3.1. Characterization of IL modified SiO₂

The amount of the target acidic IL anchored to the support surface of the SiO₂ particles was firstly quantified using elemental analysis. The elemental content was 5.81% of C, 2.13% of H and 1.141% of N, and the percentage of N in blank activated SiO₂ was measured as 0.00%. From the percentage amounts of nitrogen (N%), the weight of this was calculated to be 0.760 mmol g⁻¹ (5.268% of total weight) of the content of Im⁺·HSO₄⁻ modified on the SiO₂ surface. The FT-IR spectra of SiO₂ gel before and after modification are shown in Fig. 2(a), which were recorded between 400 cm⁻¹ and 4000 cm⁻¹. The peaks at 3448, 1636 and 1572 cm⁻¹ are attributed to O–H stretching vibration, O–H bending vibration of silanol groups and C=N stretching vibration of imidazole ring, respectively. The peaks at 1100 cm⁻¹ and 805 cm⁻¹ belong to Si–O–Si asymmetric and Si–H in-plane bending vibration, respectively. The difference of the width of the Si–O–Si vibration at 1100 cm⁻¹ before and after modification indicates that the free silanol groups have participated in the silylation process, which was also suggested by the decrease of the exclusive silanol band above 3000 cm⁻¹ in the spectrum of modified SiO₂. The signal related to the asymmetric stretching vibration of S=O usually appears at 1180 cm⁻¹, which is overlapped with the broad peak of the Si–O–Si asymmetric vibration. According to the previous analysis, it can be proven that Im⁺·HSO₄⁻ has been attached on to the surface of SiO₂ successfully. The thermal gravity and differential thermal gravity (TG-DTG) curves of SiO₂·Im⁺·HSO₄⁻ are shown in Fig. 2(b), and it is found every weight loss corresponds to the weight percentage of different components in the structure of the sorbent when they are lost in continuous heating. Specifically, the IL modified SiO₂ possesses an obvious weight loss of 5.3% in the temperature range from 350 °C to 450 °C and a strong exothermic peak at 392 °C, which agrees with the results of element analysis for the amount of immobilized IL (5.268% of total weight) and is closely related to the decomposition of IL fragment combined on the SiO₂ surface. However, the gradual mass loss in the range of 100–350 °C and 450–800 °C is attributed to the elimination of physically adsorbed water and decomposition of the silanol groups, the residual methoxyl side groups as well as the other organic fragments on the support.

Fig. 2 FT-IR spectra of KBr discs (a) and thermogravimetry-differential thermogravimetry (TG-DTG) curves of SiO₂·Im⁺·HSO₄⁻ (b).

3.2. The effects of adsorption conditions

3.2.1. Effect of initial concentration. By changing the initial concentration (50–650 mg L⁻¹) of the aqueous solution of BSA, its effect on adsorption behavior was investigated when the solid-to-liquid ratio was 50 : 25 mg mL⁻¹, temperature was 30 °C, pH was 5 and adsorption duration was 2.5 h. As shown in Fig. 3(a), it is found that the adsorption efficiency (%) will be lowered with the increase of sample concentration because the high sample concentration can enhance the squeezing effect between the BSA macromolecules. By contrast, the adsorption capacity (mg g⁻¹) could be increased by the increasing sample concentration before adsorption saturation and the adsorption capacity will reach its maximum value when the sample concentration was more than 650 mg L⁻¹. The results indicate that SiO₂·Im⁺·HSO₄⁻ has a greater capacity, however, the increasing intermolecular interaction between the BSA adsorbates is not unfavourable to their binding with the supported IL sorbent when the concentration of sample solution increases to a certain extent.

Fig. 3 Effects of initial concentration (a), solid–liquid ratio (b), pH (c) and temperature (d) on the adsorption performance.

3.2.2. Effect of solid–liquid ratio. Because previous researchers have proved that the solid–liquid ratio (w/v) is also a crucial parameter in many separation processes on various sorbents, in this research, different ratios of the mass (mg) of SiO₂·Im⁺·HSO₄⁻ to the volume (mL) of the BSA solution with an initial concentration of 650 mg L⁻¹ were investigated at 30 °C at a pH of less than 5. From Fig. 3(b), it is shown that the adsorption efficiency (%) can be improved markedly when the amount of solid–liquid ratio is increased, which should be attributed to the fact that more adsorption sites and chances are available for the target proteins. When the solid–liquid ratio was more than 3.6 mg mL⁻¹, no significant change was observed and at this time the BSA molecules in solution are adsorbed completely. Furthermore, the opposite trend is found for the change of the adsorption capacity (mg g⁻¹) with the increasing solid–liquid ratio, because the solid–liquid ratio of 0.4 mg mL⁻¹ is enough for BSA of a given amount and an excess of sorbent is useless under the investigated conditions.

3.2.3. Effect of pH values. The existing form of protein and the molecular interaction between the protein and SiO₂·Im⁺·HSO₄⁻ were determined using the pH value of the sample solution, which thereafter could affect the adsorption performance of the sorbent. Here the effect of pH in the range of 2–10 for the adsorption of BSA solution with an initial concentration of 450 mg L⁻¹ was investigated at 30 °C when the solid–liquid ratio was 50 : 25 mg mL⁻¹ and the adsorption duration was 2.5 h. As shown in Fig. 3(c), the adsorption capacity (mg g⁻¹) reaches its maximum value at a pH of less than 4.7 (isoelectric point of BSA is 4.7). Furthermore, it will decrease steeply in the range of pH < 4 because the strong acidic condition can severely damage the structure of BSA. When the pH value is above 7, the change of adsorption capacity is not obvious. A basic condition will have a certain influence on the structure of BSA, which is not as significant as that of an acidic condition.

3.2.4. Effect of adsorption temperature. Finally, the effect of temperature on the adsorption capacity of BSA by SiO₂·Im⁺·HSO₄⁻ was investigated in the temperature range of 10–50 °C (uncertainty of temperature = ±0.1 °C) when the solid-to-liquid ratio was 50 : 25 mg mL⁻¹, BSA concentration was 650 mg L⁻¹, pH was 5 and adsorption duration was 2.5 h. The experimental results are shown in Fig. 3(d), and it is found that when the temperature is rising, the adsorption capacity decreases slowly, which can indicate that the adsorption process is exothermic and adsorption should be carried out under a lower temperature. An excessive temperature will result in the aggregation, denaturation or partial hydrolysis of protein. Compared with the three former factors, the adsorption temperature has a relatively weaker effect on the adsorption process. The detailed research on the adsorption thermodynamics is discussed in Section 3.4.3.

Under the previously described investigated conditions, the maximum adsorption capacity of BSA on SiO₂·Im⁺·HSO₄⁻ could reach 246.5 mg g⁻¹, which is higher than that on HmIm⁺·Br⁻@Fe₃O₄ nanoparticles (182.0 mg g⁻¹),¹⁰ Cibacron Blue F3GA attached chitosan microspheres (108.7 mg g⁻¹),¹⁶ magnetic hydrogel beads based on PVA/sodium alginate/LAPONITE® RD (127.3 mg g⁻¹)¹⁷ and molecularly imprinted polymer on cadmium sulfide quantum dots (226.0 mg g⁻¹).¹⁸ Further improvements can be realized using orthogonal experiments or response surface optimization.

3.3. Adsorption mechanism

3.3.1. Adsorption kinetics. The adsorption kinetics can be investigated using the effect of time on adsorption. Fig. 4(a) shows the adsorption kinetics curve of SiO₂·Im⁺·HSO₄⁻ for target protein solutions with different initial concentrations (including 250, 350 and 450 mg L⁻¹) at 30 °C. The first 30 min, was the stage of initial binding or anchorage on the sorbent surface and the uptake of BSA increased quickly, once the BSA molecules were bound into the mesoporous structure of the IL modified SiO₂ gel and combined with deeper active sites, so the kinetics curve gradually became horizontal. Obviously, the first step of adsorption was more rapid than the second one, and it took 2 h to reach complete adsorption equilibrium at which point no more adsorption was observed. In order to explore the adsorption mechanism of SiO₂·Im⁺·HSO₄⁻ for the protein solute, pseudo-first-order kinetic and pseudo-second-order kinetic models were used to correlate all of experimental data according to a previous report¹⁹ (see ESI material 1†), and the adsorption kinetic parameters (k₁, k₂ and q_e) are summarized in Table 1. It is clear that the R² of the second-order model is above 0.9900 and the matching degree between the calculated adsorption amount at equilibrium (q_e,cal) and the experimental data was ideal when this model was used. These results indicate that the adsorption rate is determined by hybrid mechanisms including chemical interaction between BSA and SiO₂·Im⁺·HSO₄⁻. However, the blank SiO₂ gel can only provide the single physical adsorption before it is modified with IL. Therefore, the pseudo-second-order model was more suitable than the pseudo-first-order model to describe the adsorption kinetic behavior and parameters. The initial high sorption rate was possibly controlled by film diffusion and electrostatic effect, and then intraparticle diffusion resulted in a lower rate at the second stage.²⁰

Fig. 4 Adsorption kinetics curves (a) and isotherms (b) of BSA onto SiO₂·Im⁺·HSO₄⁻.

Table 1 Parameters of pseudo-first-order and pseudo-second-order kinetic models

C0 (mg L^−1)	qe,exp (mg g^−1)	Pseudo-first-order model			Pseudo-second-order model
		k1 (min^−1)	qe,cal (mg g^−1)	R^2	k2 (g mg^−1 min^−1)	qe,cal (mg g^−1)	R^2
250	102.9600	1.4161	5.3324	0.9406	0.0884	106.3830	0.9993
350	143.0000	1.8279	6.6333	0.9603	0.0578	147.0588	0.9983
450	159.6400	2.1427	5.7448	0.9519	0.1281	161.2903	0.9995

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3.3.2. Adsorption isotherms. The adsorption isotherm curve [see Fig. 4(b)] was investigated under certain temperatures to describe the relationship between the equilibrium concentrations of the BSA molecules in solid and liquid phases for the adsorption process occurring at the two-phase interface. As two of the most widely used equations for solid–liquid systems, the Langmuir and Freundlich isotherm models are usually considered in the exploration for separation mechanism of IL.¹⁴ In our study, the adsorption isotherms of several initial concentrations (50–500 mg L⁻¹) were observed at different temperatures (20 °C, 30 °C and 40 °C) and it was found that the results were consistent with the previous research discussed in Section 3.2.4 for the effect of adsorption temperature. Obviously, a high temperature was not in favor of the exothermic adsorption process. The equilibrium adsorption capacity will increase with the increasing equilibrium concentration until adsorption saturation, and all equilibrium adsorption capacity was lowered with increasing temperature. The values of the Langmuir and Freundlich constants are listed in Table 2, and related R² were calculated from the linear regression. As shown in ESI material 2,† the experimental data fit with the Langmuir model (R² > 0.980) much better for all the tested temperatures and the Freundlich model was not suitable to describe the isotherm curve for its lower R² < 0.810. So the Langmuir model was used for understanding the adsorption process of BSA on SiO₂·Im⁺·HSO₄⁻, and this conclusion is similar to that obtained with a previous study.¹⁰ Under the investigated conditions, adsorption capacity is also controlled by pore volume, which is mainly the result of volume filling.

Table 2 Parameters of Langmuir and Freundlich models

T (°C)	Langmuir equation			Freundlich equation
	qm (mg g^−1)	Kl (L mg^−1)	R^2	Kf	n	R^2
20	139.28	0.3102	0.9989	50.2801	4.4597	0.8143
30	166.95	0.0535	0.9838	18.5191	2.2020	0.8079
40	136.80	0.5779	0.9995	62.1276	5.9207	0.7981

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3.3.3. Adsorption thermodynamics. To further explore the nature of the adsorption process, investigation of the change in values of enthalpy (ΔH), entropy (ΔS) and Gibbs free energy (ΔG) is necessary, especially for those complex adsorbates such as proteins. In this research they were calculated according to the method given in ref. 21 and the results are shown in Table 3. As can be seen, the negative values of ΔH for different initial concentrations and temperatures still suggest that it is an exothermic process for the binding of BSA onto SiO₂·Im⁺·HSO₄⁻, which is in agreement with previous findings. Furthermore, the range of ΔH from −92.451 kJ mol⁻¹ to −23.022 kJ mol⁻¹ proves that there is a strong electrostatic interaction and the process is also controlled by chemical adsorption as well as physical adsorption.²² The positive values of ΔS within the tested temperature range indicate that the randomness will increase on the liquid–solid interface during the adsorption process. This result agrees with the theory of displacement model of adsorption²³ and the adsorption process of BSA is accompanied by the desorption of physically adsorbed water. Finally, the values of ΔG in Table 3 are negative in the concentration range of 100–300 mg L⁻¹ from 293 K to 313 K, so the adsorption process can be proved to be spontaneous and thermodynamically favorable.

Table 3 Thermodynamic parameters for the adsorption of BSA on SiO₂·Im⁺·HSO₄⁻

C0 (mg L^−1)	ΔH (kJ mol^−1)	ΔS (J mol^−1 K^−1)	ΔG (kJ mol^−1)
			293 K	303 K	313 K
100	−92.451	280.759	−7.79197	−4.2271	−10.154
150	−67.466	202.519	−6.05324	−3.96949	−8.32385
200	−47.473	126.883	−5.01424	−3.62535	−6.46643
250	−24.572	65.399	−4.26105	−4.18228	−5.8585
300	−23.022	64.805	−3.83613	−2.39388	−3.91403

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3.3.4. Characterization and comparative study for mechanism. Besides the adsorption kinetics, isotherms and thermodynamics, some experiments were employed to further discover the adsorption mechanism of BSA on SiO₂·Im⁺·HSO₄⁻. First of all, the morphology of the sorbent particles was observed before and after adsorption, using SEM and TEM. As shown in Fig. 5, the surface is porous and the pore size is relatively uniform before adsorption, however, because of the porous structure of the sorbent surface, light is easily transmitted and the color of the particle edge is relatively lighter. After adsorption, surface pores on SiO₂·Im⁺·HSO₄⁻ have been filled with BSA, and the light transmission is reduced because of the decrease of the number of uncovered pores. This phenomenon proves the existence of macroporous adsorption in the hybrid mechanism.

Fig. 5 SEM photos (×200) before adsorption (a) and after adsorption (b) together with TEM photos before adsorption (c) and after adsorption (d).

In order to investigate the specific surface area and pore size distribution of the sorbent, N₂ adsorption–desorption was selected as the most effective method. Before adsorption of protein, N₂ adsorption–desorption of SiO₂·Im⁺·HSO₄⁻ showed an obvious type IV isotherm curve, which had typical characteristics of a mesoporous adsorbent with an H2-type hysteresis loop. According to the Brunauer–Emmett–Teller adsorption equation for specific surface area analysis,²⁴ the result in ESI material 3† shows there are four major signal peaks within 20 min. The first negative signal is the adsorption peak of N₂ on the sample. After that, stepwise desorption by N₂ for the sample begins. The second peak (76.2 m² g⁻¹) around 400 s is attributed to the removal of standard material (SiO₂), and then the third peak (211.3 m² g⁻¹) near 750 s belongs to SiO₂·Im⁺·HSO₄⁻ before adsorption, which proves that the surface of the IL modified SiO₂ gel has a porous structure and its specific surface area is about three times that of the standard material. Finally, the peak (124.4 m² g⁻¹) at 995 s corresponds to BSA-bound sorbent, which also indicates that the pores are filled with protein and the specific surface area decreases.

Zeta potential analysis was also performed, using a PALS version 3.43 analyzer (Brookhaven Instruments, Austin, TX, USA) to characterize SiO₂·Im⁺·HSO₄⁻ before and after adsorption at a pH of less than 4. Specifically, 5 mg of the samples to be tested were ground and then suspended in 5 mL of deionized H₂O, and then the supernatant was analyzed after high speed centrifugation. The zeta potential of the sorbent varied from 4.05 mV to 5.25 mV when adsorption was completed. This could be interpreted as proof of the incorporation of BSA, which is attributed to the electrostatic interaction between the protein and sorbent.^25,26 As is known, BSA will have a positive charge when the pH is lower than its isoelectric point (PI = 4.7). As a function of the surface charge of sorbent particles, the zeta potential will increase as the active sites become occupied by the protein molecules with positive charge. Obviously, the main interaction occurs between the HSO₄⁻ anions of the sorbent and various nitrogen groups with positive charge in arginine, histidine, lysine or tryptophan residues on the peptide chains of BSA.

Finally, two comparative experiments were designed to further understand the mechanism. Four other sorbents [benzothiazolium hexafluorophosphate-supported silica (SiO₂·Bth⁺·PF₆⁻), imidazolium hexafluorophosphate-supported silica (SiO₂·Im⁺·PF₆⁻), imidazolium chloride-supported silica (SiO₂·Im⁺·Cl⁻), and imidazolium hexaborophosphate-supported silica (SiO₂·Im⁺·BF₆⁻)] were synthesized according to a method reported in a previous study,¹⁴ and their adsorption performance was compared with SiO₂·Im⁺·HSO₄⁻ together with blank SiO₂ under the same conditions. The adsorption capacity of four proteins with various PIs (BSA: 4.7, hemoglobin: 6.9, papain: 8.75, protamine sulfate: 12.4) was compared on SiO₂·Im⁺·HSO₄⁻ under different pHs. As shown in Fig. 6(a), blank SiO₂ has the lowest adsorption capacity, which will increase when the surface is modified with IL. Furthermore, for water soluble protein such as BSA, IL with hydrophilic anions (HSO₄⁻, BF₆⁻, Cl⁻) are better than hydrophobic ones with PF₆⁻ when their cations are same. The Im IL is better than the Bth IL with the same anion for its smaller cation and less steric hindrance. Of the four anions tested, HSO₄⁻ has the highest acidity, which can provide a strong interaction with alkaline groups on BSA through electrostatic attraction. On the basis of the results shown in Fig. 6(b), all of four different proteins reach their maximum adsorbed amount on SiO₂·Im⁺·HSO₄⁻ when the pH value is around that of their PIs, and this finding is very similar to results reported in a previous paper.²⁷ Under this condition these proteins are in a state of contraction, which is conducive to their adsorption in the form of inclusion by the immobilized IL. As a useful supplement, the adsorption capacity for three proteins with similar PIs [BSA: 4.7, angiotensin-converting enzyme (ACE): 4.9 and collagen type V: 4.8] was investigated [see Fig. 6(c)]. It can thus be concluded the adsorption capacity will decrease with the increase of protein volume (ACE: 60 kDa, BSA: 66 kDa, collagen type V: 270 kDa). However, collagen type V has very poor solubility in water, which is not beneficial for its complete contact with SiO₂·Im⁺·HSO₄⁻. The previously described experiments prove the existence of a hybrid mechanism and again, the adsorption includes physical and chemical processes.

Fig. 6 Comparison of adsorption capacity for different sorbents (a), proteins with various PIs (b) and proteins with similar PIs (c).

3.4. Desorption and reusability study

In the previous study, the properties of a solvent have been found to have an important impact on the desorption process. Here different elution reagents were preliminarily screened (including methanol, ethanol, acetone, sodium dihydrogen phosphate and NaCl aqueous solution). Considering the fact that acid and alkaline easily exert their influence on the structure of proteins and result in inconvenience during post-processing, they were abandoned in the experiment. As a result, it was found NaCl–H₂O gave the best performance, and could desorb BSA from the sorbent through competitive adsorption. Furthermore, when the SiO₂·Im⁺·HSO₄⁻ particles were washed with NaCl–H₂O, the eluent showed a negative result with the precipitation reaction of barium chloride reagent after being neutralized, which indicated no HSO₄⁻ could be replaced by Cl⁻ and the related anion-exchange process in the last step during the preparation of IL modified SiO₂ was non-reversible. In the following study, aqueous solutions of NaCl with different concentrations (0–2 mol L⁻¹) were tested and the results of their performance is shown in Fig. 7(a). When a 1.5 mol L⁻¹ (6%) aqueous solution NaCl was used, the highest desorption efficiency of 94.9% could be obtained, and then SiO₂·Im⁺·HSO₄⁻ was washed using deionized H₂O to neutralize it and then it was dried at 60 °C under vacuum before the next adsorption. If the sorbent is used in a column for solid-phase extraction, it can be directly reused without drying and repacking. From the result shown in Fig. 7(b), the majority can be desorbed within 30 min, and after that the desorption rate gradually changes from fast to slow. Finally, it was found the adsorption efficiency of SiO₂·Im⁺·HSO₄⁻ still reached 89.58% in the fifth run so it could be recycled and reused several times. The obvious color and status changes of the IL modified SiO₂ particles were not observed, and the maximum UV absorbance wavelength of BSA was still 278 nm after desorption. After measurement of circular dichroism, the positive and negative peaks at 192 nm (+), 208 nm (−) and 222 nm (−) were still observed in the desorbed BSA. These results prove that no denaturation or degradation was found.

Fig. 7 Effects of NaCl concentration (a) and time (b) on desorption efficiency.

3.5. Selective adsorption of BSA and BHb

According to the conclusions drawn in Section 3.2.3, pH has a significant influence on the adsorption capacity, which makes the selective adsorption for proteins with different PIs become possible. BSA and BHb are homologous proteins from cow blood, and they have similar molecular weights (BSA: 66.4 kDa, BHb: 64.5 kDa) and different PIs (BSA: 4.7, BHb: 7.0). Experiments to achieve their selective adsorption by the adjustment of pH were tried, and the result is shown in Fig. 8(a). When the pH is lower than 3, the strong acidic environment makes the proteins denaturate and the adsorption capacity decreases. The adsorption capacity for BSA (134.3 mg g⁻¹) and BHb (69.0 mg g⁻¹) reaches the maximum level at their PIs, and their adsorbed amounts have the greatest difference at pH 5. So a sample composed of a mixture of the two equivalent proteins was investigated and their aqueous solution was adsorbed by SiO₂·Im⁺·HSO₄⁻ at pH 5. The UV-vis absorbance of the sample mixture at 278 nm belongs to BSA and the absorbance at 406 nm is attributed to BHb, and their contents were determined according to the absorbance at the two wavelengths (the standard curve of BSA is y = 0.0164x + 0.0486, R² = 0.9996; the standard curve of BHb is y = 0.0116x − 0.0003, R² = 0.9999) respectively. Fig. 8(b) shows the result of selective adsorption and their desorption capacities are a little different to that of the individual adsorptions. The adsorption capacity for BSA in the mixture is 116.1 mg g⁻¹, which is 87% of the adsorption capacity of BSA alone, and the adsorption capacity for BHb increases from 20.1 mg g⁻¹ to 26.3 mg g⁻¹. By a study of adsorption kinetics for the two proteins, it is found that the adsorption rate of BHb is higher than that of BSA, and the former can occupy the active sites rapidly and make the adsorption capacity of the latter decrease during competition. Finally, the actual sample of cow blood was tested in the selective adsorption. The sample which was diluted with deionized H₂O, was filtered and adjusted to pH 5, successively. A little shift of absorbance wavelength of the two proteins was found because of other co-existing substances in the blood sample. On the basis of the data in Fig. 8(c), the adsorption capacity for BSA is 144.0 mg g⁻¹ and that for BHb is 29.5 mg g⁻¹. After the elution with 1.5 mol L⁻¹ (6%) NaCl aqueous solution, a purity of 91.2% and yield of 27.8% (weight of BSA/weight of cow blood × 100%) was achieved for BSA.

Fig. 8 Adsorption capacity of BSA and BHb under different pHs (a) and selective adsorption results for the samples of their mixture (b) together with actual cow blood (c) on SiO₂·Im⁺·HSO₄⁻.

4. Conclusions

In the present work, SiO₂·Im⁺·HSO₄⁻ was synthesized, characterized and evaluated for the adsorption and separation of BSA. The effects of major operating conditions including adsorption temperature, initial sample concentration, solid–liquid ratio and pH were all investigated. The adsorption data fit well with the Langmuir model and the adsorption kinetics were described well by a pseudo-second-order model. It was proved by the thermodynamic study that the adsorption of BSA onto SiO₂·Im⁺·HSO₄⁻ was a spontaneous and exothermic process, which was controlled by chemical and physical adsorption. The adsorption mechanism was also investigated using detailed characterization and a comparative study. In addition, the desorption conditions and reusability of SiO₂·Im⁺·HSO₄⁻ were also studied, and results showed that it could be recycled several times and no denaturation or degradation of protein was found after desorption. In the optimal pH environment, the selective adsorption of BSA and BHb can be successfully realized. In summary, IL modified SiO₂ can be considered as a satisfactory sorbent for application in the fields of small molecules together with proteins. With a promising performance in the processes of adsorption and desorption, it can be expected to play a greater role in the separation of similar biological macromolecules.

Acknowledgements

Preparation of this paper was supported by National Natural Scientific Foundation of China (No. 81373284) and 2013 Scientific Research Foundation of Sichuan University for Outstanding Young Scholars.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra23372d

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